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Extracellular Calcium and cAMP: Second Messengers as "Third Messengers"?


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Calcium and cyclic AMP are familiar second messengers that typically become elevated inside cells on activation of cell surface receptors. This article will explore emerging evidence that transport of these signaling molecules across the plasma membrane allows them to be recycled as "third messengers," extending their ability to convey information in a domain outside the cell.
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It seems that yet another domain within the organism
touched by Ca
and cAMP is the extracellular space. It
has long been known that intracellular signaling events
are associated with changes in second messenger con-
centrations outside the cell. Can these fluctuations be
regarded as “signals” in their own right? This article will
address how cAMP and Ca
move across the cell mem-
brane, the potential mechanisms for sensing these
extracellular changes in messenger concentration, and
the physiological outcomes of these signaling events.
Export of cAMP from
Stimulated Cells
In 1963, just 5 years after the seminal descriptions of
the second messenger function of cAMP, Davoren and
Sutherland reported the existence of a probenecid-
sensitive mechanism for cAMP extrusion in nucleated
pigeon erythrocytes (18). Over the years, numerous
reports have appeared demonstrating that cAMP can
be expelled following agonist stimulation from a wide
variety of cell types, including adipocytes (57), hepato-
cytes, renal epithelial cells (56), neuronal cells (53),
fibroblasts, T lymphocytes (67), and skeletal muscle
(27). In some earlier studies, investigators even used
determinations of external [cAMP] as a surrogate for
measurements of hormone-dependent cAMP accu-
mulation inside the cell (8). Nevertheless, this ability
of cells to eject the second messenger is apparently
cell-type specific, since certain other cell types do not
appear to extrude cAMP whatsoever.
cAMP export can proceed across a concentration
gradient, is temperature dependent, and requires ener-
gy in the form of ATP (FIGURE 1). This process is sus-
ceptible to inhibitors of organic anion transport, such
as probenecid and sulfinpyrazone (56). In a colonic
carcinoma model (CC531
), the efflux of cAMP was
found to be exquisitely sensitive to cell swelling (28).
Certain multi-drug resistance proteins in the MRP fam-
ily have now been identified as significant pathways for
cAMP egress (39, 69). These proteins belong to the
superfamily of ATP binding cassette (ABC) proteins,
specifically the ABCC subfamily. Most of these are
known to function as active membrane transporters for
organic anions and various drugs [e.g., for bile acids
and chemotherapeutic agents, including nucleoside
1548-9213/05 8.00 ©2007 Int. Union Physiol. Sci./Am. Physiol. Soc.
The foundations of the second messenger concept were
established nearly 50 years ago when Earl Sutherland
and Ted Rall identified a heat stable factor that mediat-
ed the intracellular actions of the hormones glucagon
and epinephrine on glycogen metabolism in the liver
(49). That factor was, of course, cyclic AMP, a discovery
that earned Sutherland a Nobel Prize in 1971 (58).
The next signaling molecule to be officially consid-
ered as a second messenger was the calcium ion (12).
The importance of Ca
as a trigger of muscle contrac-
tion was known since the 19th century from the pio-
neering work of London-based physiologist Sydney
Ringer. However, acceptance of the general signal
transduction function of Ca
, as originally proposed
by Lewis Victor Heilbrunn in the 1940s, initially met
with resistance (43). Recognition of the validity of this
theory slowly built following the identification of the
ATPases, intracellular Ca
release channels, and
protein targets for the Ca
signal throughout the 1960s
and 1970s. Around this same time, several laboratories
managed to make direct measurements of agonist-
stimulated intracellular Ca
transients using the lumi-
nescent jellyfish photoprotein aequorin. Together,
these findings firmly vaulted Ca
to the status of a
bona fide second messenger.
and cAMP are now recognized as universal reg-
ulators of cell function. Between them, these two clas-
sic textbook second messengers impact nearly every
aspect of cellular life in diverse organisms ranging
from amoebae to plants to human beings. A recurrent
theme that has emerged since the early descriptions of
these fundamental messengers is the importance of
localized signaling events within the cell (6, 16, 60, 70).
By confining Ca
or cAMP to precise subcellular
domains (e.g., plasma membrane, organelles), these
molecules can selectively activate a subset of targets,
thereby expanding the repertoire and range of the sig-
nal. Discovery of this property was greatly facilitated
by the parallel development of fluorescent probes and
digital microscopic imaging techniques, methods ini-
tially applied for the visualization of Ca
events (71). More recently, FRET-based indicators for
imaging cAMP in single cells have been used to con-
firm that the metabolism and disposition of cAMP can
be regulated independently in different parts of the
cell (25, 42, 46).
Extracellular Calcium and cAMP: Second
Messengers as “Third Messengers”?
Aldebaran M. Hofer and
Konstantinos Lefkimmiatis
Department of Surgery, VA Boston Healthcare System and
Brigham & Women’s Hospital, Harvard Medical School,
West Roxbury, Massachusetts
Calcium and cyclic AMP are familiar second messengers that typically become ele-
vated inside cells on activation of cell surface receptors. This article will explore
emerging evidence that transport of these signaling molecules across the plasma
membrane allows them to be recycled as “third messengers,” extending their abili-
ty to convey information in a domain outside the cell.
PPHHYYSSIIOOLLOOGGYY 2222:: 332200332277,, 22000077;; 1100..11115522//pphhyyssiiooll..0000001199..22000077
analogs; see Kruh and Belinsky for review (39)]. In par-
ticular, MRP4, MRP5, and MRP8 are established trans-
porters of cyclic nucleotides (cAMP and cGMP) (69).
These drug efflux pumps often assume polarized distri-
butions in epithelial cells (e.g., MRP4 is found in the
apical membrane of kidney tubule cells and the baso-
lateral membrane of prostate glandular cells) (39).
Quantitatively, the amount of cAMP expelled by cer-
tain cell types can be quite significant. For instance,
human CD4
T lymphocytes stimulated with cholera
toxin release more than 50% of the total cAMP pro-
duced over a 24-h period to the extracellular space
(67). Strewler found an even more vigorous degree of
probenecid-sensitive export in polarized LLC-PK
renal epithelial cells; 40 min after stimulation with
arginine vasopressin, the external cAMP concentra-
tion in the apical bath was more than twice that
retained inside the cells (56). In humans, the concen-
tration of cAMP in plasma and urine can become dra-
matically elevated under a variety of physiological
conditions and also following infusion of exogenous
cAMP-elevating hormones (particularly epinephrine,
parathyroid hormone, and glucagon). For example,
Hendy and colleagues showed many years ago that
infusion of human subjects with a large bolus of
glucagon causes massive release of cAMP from the
liver, elevating plasma levels from low nanomolar lev-
els to over 450 nM within 10 min (31). This could
translate into dramatic local accumulations of cyclic
nucleotides in the diffusion-restricted spaces adjacent
to cells. Direct measurements using microdialysis
techniques have further demonstrated that significant
fluctuations in extracellular cAMP can occur in intact
brain tissue during agonist treatment (11).
The cAMP extrusion mechanism has been largely
dismissed as a means of regulating the intracellular
cAMP signal, since the cyclic nucleotide phosphodi-
esterases (PDEs: the enzymes that degrade cAMP and
cGMP) already provide powerful and rapid control of
intracellular cAMP (3, 39). Furthermore, it has been
argued that this strategy is too energetically unfavor-
able to be a viable means of regulating the cyclic
nucleotide levels within the cell, with the further
adverse consequence of depleting the cellular purine
pool (38). As seen in FIGURE 2, however, acute inhibi-
tion of this efflux pathway with probenecid can result in
measurable increases in resting intracellular [cAMP] as
measured using sensitive FRET-based reporters. This
suggests that in some cells this molecular device may
be capable of altering resting cAMP levels and, poten-
tially, the profile of the intracellular cAMP signal (27,
56), in addition to altering extracellular levels of cAMP.
Actions and Targets of
Extracellular cAMP
In the social amoeba Dictyostelium discoideum, extra-
cellular cAMP serves as a chemical alarm bell that
signals single-celled individuals to aggregate during
stress into a multicellular “slug” or pseudoplasmodi-
um. This fascinating organism, long used as a model
for cellular migration and chemotaxis, is known to
express four G-protein-coupled receptors (GPCRs)
used to detect secreted cAMP (cAR1, cAR2, cAR3,
cAR4; not to be confused with the Ca
-sensing recep-
tor “CaR” described below) (5). These receptors have
affinities for cAMP ranging from sub-micromolar to
To date, no mammalian homologs of this well char-
acterized cAMP receptor have been reported. It is
noteworthy, however, that the Dictyostelium GPCRs
share some limited sequence similarity to the secretin
family of GPCRs expressed in mammalian cells,
which includes receptors for parathyroid hormone
and calcitonin. It has been speculated that the latter
class of GPCRs may mediate the actions of extracellu-
lar cAMP in vertebrates (5). Extracellular cAMP is
known to have many actions on diverse organ
PHYSIOLOGY • Volume 22 • October 2007 •
FIGURE 1. “Third messenger” activity of extracellular cAMP
Intracellular cAMP is typically generated via adenylyl cyclases (AC) fol-
lowing hormonal activation of G-protein-coupled receptors (GPCRs)
linked to the heterotrimeric G-protein, G
. Once formed, the second
messenger can be actively transported to the extracellular space via a
probencid- and sulfinpyrazone-sensitive efflux mechanism belonging to
the ATP-binding cassette (ABC) transporter family. Extracellular cAMP is
hypothesized to have direct actions on putative receptor proteins (as of
yet unidentified) that are expressed on neighboring cells. Alternatively,
it is well established that extracellular cAMP can be sequentially metab-
olized, first by ecto-phosphodiesterase to adenosine monophosphate
(AMP), and then by ecto-5’-nucleotidase to adenosine. Adenosine can
then act as a paracrine or autocrine messenger to activate other signal
transduction cascades via one of four subtypes of adenosine receptors
, A
, A
, A
). In addition, since cAMP is relatively stable in blood,
circulating cAMP can be converted by ecto-enzymes at a distant site,
effectively rendering cAMP as a prohormone for adenosine (which has a
fleeting half-life in the circulation).
cell model (22). Again, intracellular cAMP and extra-
cellular cAMP had opposing actions on PGHS-2
expression, causing upregulation and downregulation
of the enzyme, respectively.
Paracrine Action of cAMP on the
Renal System: The Extracellular
cAMP-Adenosine Pathway
The hormone glucagon, which is released from the
pancreas directly into the portal blood flow, causes
intracellular cAMP signaling in hepatocytes, and, as
suggested above, this leads to substantial efflux of
cAMP into the general circulation. It appears that an
important target of this circulating cAMP is the renal
system, particularly the proximal tubule. Glucagon is
well known to cause a marked enhancement of renal
sodium and phosphate excretion in vivo, although
specific receptors for glucagon have never been iden-
tified in the kidney. This prompted Bankir and
colleagues (5) and others to propose that cAMP
released from the liver might be acting as a
circulating factor mediating the renal actions of
glucagon. A sequence of studies by Ahloulay et al.
showed that cAMP infusion alone reproduced the
actions of glucagon on renal Na
and PO
(2). This phenomenon has been named the
“pancreato-hepatorenal cascade.
A comprehensive series of animal experiments car-
ried out by Jackson and coworkers (36, 37) have given
a further twist on this general theme. These investiga-
tors showed that the cAMP entering the general
circulation from the liver is able to undergo enzymatic
conversion to adenosine once it reaches the kidney
(FIGURE 1). Adenosine has a short half-life in the cir-
culation (~1 s); therefore, cAMP (which is stable in
plasma) may be regarded as a sort of prohormone for
adenosine. Once produced (either locally or at a dis-
tant site), adenosine can activate one of four different
receptor subtypes (A
, A
, A
, and A
). Complex sce-
narios can be envisioned considering that adenosine
receptor subtypes A
and A
interact with G
reduce intracellular cAMP levels in target cells, where-
as the A
and A
subtypes serve to increase cAMP via
. Therefore, cAMP released from one cell type could
conceivably initiate cAMP signaling in a neighboring
cell or suppress cAMP signaling depending on the par-
ticular adenosine receptor subtype expression pattern.
As described above, the cAMP-adenosine pathway
is prominent in the kidney, but substantial evidence
for this phenomenon also exists in the central nervous
system (11, 21, 38). Moreover, the cAMP-adenosine
pathway has been speculated to be important in the
cardiovascular system and also for systemic metabolic
homeostasis (57). The presence of the pathway does
not preclude the possibility that cAMP may exert
direct actions on cells in addition to indirect effects via
adenosine production.
PHYSIOLOGY • Volume 22 • October 2007 •
systems, including renal, hepatic, and central nervous
system. This subject has recently been reviewed in depth
by Bankir et al. (5) and Jackson and Raghvendra Dubey
(38). As described below, some of these actions may be
the indirect result of the metabolism of cAMP to adeno-
sine in the extracellular space (the “extracellular cAMP-
adenosine pathway”), although some effects appear to
be direct. For example, Sorbera and Morad (55) showed
in 1991 that 50 M extracellular cAMP rapidly (~50 ms)
inhibited a sodium current in ventricular myocytes
derived from several vertebrate species. This effect was
sensitive to pertussis toxin, suggesting a GPCR-based
mechanism dependent on G
or G
proteins (55). As
another example, secreted cAMP (but not adenosine)
derived from stimulated human CD4
T lymphocytes
was recently shown to exert significant growth effects on
neighboring T cells in a co-culture system (67).
Detrick and colleagues demonstrated that nanomo-
lar concentrations of extracellular cAMP and cGMP
(but not adenosine or guanosine) enhanced colony
formation in myeloid progenitor cells. Interestingly,
membrane permeant forms of the cyclic nucleotides
used at high micromolar concentrations had the
opposite effect on the proliferation of the cells, imply-
ing that an intracellular elevation of cAMP or cGMP
could antagonize the action of extracellular second
messenger (20). Elalamy and colleagues have provid-
ed compelling evidence for the involvement of an
ecto-PKA (protein kinase A) in mediating the actions
of extracellular cAMP (used at a concentration of 5
M) on expression of prostaglandin H synthase
(PGHS-2) in a pulmonary microvascular endothelial
FIGURE 2. Blockade of cAMP extrusion with probenecid alters
intracellular free [cAMP]
Intracellular cAMP was imaged using a FRET-based biosensor (46) in sin-
gle HEK293 cells as described previously (25). This sensor (courtesy of Dr.
Kees Jalink) is based on the cAMP binding protein Epac, which has been
labeled with CFP and YFP. cAMP-dependent conformational changes of
the Epac protein result in changes in FRET, providing a measure of free
[cAMP]. Acute treatment of cells with 1 mM probenecid caused a small
but significant change in the resting FRET signal (the 480:535 nm emis-
sion ratio), consistent with an increase in intracellular [cAMP]. These data
suggest that the cAMP export process can contribute to intracellular
cAMP homeostasis, in addition to mediating the elevation in extracellular
cAMP. Shown for comparison is the action of the direct adenylyl cyclase
activator forskolin (50 M).
Extracellular Calcium as a Third
Just as it has long been known that intracellular cAMP
signaling events are associated with extracellular
accumulation of the second messenger, so has it been
long appreciated that Ca
can fluctuate outside cells,
owing to activation of influx and efflux pathways for
the cation during Ca
signaling events (41). As with
cAMP, early measurements of hormone-stimulated
signals frequently relied on determinations of
in the external media. Because diffusion is great-
ly limited in the interstitial spaces (which occupy only
a fraction of the tissue volume; e.g., ~20% in brain
tissue) (66) and the buffering capacity for Ca
the cell is so much greater than outside, these fluxes
can lead to significant alterations in free [Ca
] in the
extracellular milieu.
Fluctuations in Extracellular Ca
As indicated in FIGURE 3, agonist-stimulated Ca
signaling events involve 1) the release of Ca
internal storage compartments into the cytoplasm
via intracellular release channels (i.e., the InsP
receptor); 2) the extrusion of Ca
into the extracellu-
lar space by plasma membrane Ca
ATPases (PMCA)
or other export mechanisms (e.g., Na
ers); and 3) the activation of Ca
entry through
store-operated channels (SOCs), such as the recently
identified Ca
release-activated pathways known as
the Orai proteins (47).
Tepikin and colleagues have provided direct
demonstrations of the significant quantitative impact
of the Ca
extrusion process on extracellular Ca
levels adjacent to stimulated cells (61–63). One study
employed simultaneous real-time measurements of
intracellular and extracellular [Ca
] in single pancre-
atic acinar cells suspended in a small droplet (40–90
times the volume of the cell; FIGURE 4). By measuring
the extracellular [Ca
] in the droplet with a Ca
sitive dye, it was estimated that the total intracellular
calcium content was reduced by 0.7 mM during
cholinergic stimulation, owing to active transport of
the cation by the PMCA.
Temporal and spatial separation of Ca
entry and
efflux across the plasma membrane can give rise to
physiologically significant excursions in extracellular
] (13, 35), particularly in polarized epithelial cells
and other functionally polarized cells such as neurons
(33). For example, Caroppo and colleagues showed
that [Ca
] in the luminal micro-compartment of the
intact gastric gland increases by 200–500 M following
cholinergic stimulation, owing to an abundance of
PMCA on the apical membrane of the gastric
epithelial cells (13). At the same time, a comparable
depletion of Ca
was recorded in the basolateral inter-
stitium of the intact mucosa as a result of Ca
via pathways located predominantly at the basal cell
side. As described below, these extracellular [Ca
fluctuations have been shown to have functional con-
It is also well established that specific elements of the
-handling machinery (as well as certain Ca
sors) can be confined in cell surface microdomains,
such as caveolae (17, 26), potentially giving rise to local
gradients of Ca
in the caveolar nanospaces. Other fac-
tors can influence free [Ca
] in the external milieu,
including dilution and concentration of ionic species,
owing to cellular water transport. In addition, the
PHYSIOLOGY • Volume 22 • October 2007 •
FIGURE 3. “Third messenger” activity of extracellular Ca
Local extracellular [Ca
] can fluctuate as a consequence of agonist-
induced intracellular Ca
signaling events. In the typical scenario, acti-
vation of a G
-coupled receptor by a Ca
-mobilizing agonist results
in inositol 1,4,5-trisphosphate (InsP
) production, giving rise to the lib-
eration of stored Ca
via InsP
receptor release channels in intracellular
pools. A substantial fraction of Ca
released into the cytoplasm is
rapidly extruded by plasma membrane Ca
ATPases (PMCAs), poten-
tially resulting in significant local elevation of the extracellular [Ca
Store emptying also triggers Ca
influx via store-operated Ca
nels (SOCs) in the plasma membrane, leading to transient depletion of
in the volume-limited interstitial spaces. The ensuing local fluctua-
tions in Ca
can influence a variety of Ca
-sensing proteins on adja-
cent cells or on the same cell. Examples include HERG K
several types of nonselective cation channels, and a host of G-protein-
coupled receptors modulated by extracellular Ca
(e.g., the extracellu-
lar Ca
-sensing receptor, the GPRC6A orphan receptor, or
metabotropic glutamtate receptors). Gap-junction hemichannels and
the transmembrane protein notch are also susceptible to alterations in
extracellular [Ca
] (32).
Extracellular Ca
Although specific GPCRs for cAMP have not been
identified in mammalian cells, cell-surface receptors
for Ca
are known to exist, and some of these have
been well characterized. Without question, the best
known of these is the extracellular calcium-sensing
receptor (CaR), which was originally cloned from
bovine parathyroid gland in 1993 by Brown and col-
leagues (9). The structural and functional properties
of this widely expressed divalent cation receptor have
been reviewed extensively elsewhere (10, 33) and will
not be addressed in detail here. The CaR (of which
only a single isoform appears to exist) is indispensa-
ble for life in mammals, acting as the Ca
sensor that
controls systemic Ca
and Mg
homeostasis via PTH
secretion. An emerging literature describes numer-
ous physiological functions of this receptor through-
out the body and in diverse vertebrate species,
including birds and fish. Deletion of CaR is lethal, but
the developments of viable “rescued” CaR knockout
mouse models that maintain normal parathyroid
function are being used increasingly to examine this
receptor’s physiological role in other organ systems
(1, 45).
CaR is a member of family C of the GPCR superfam-
ily, which also includes three taste receptors (T1–T3),
the GABA
receptors, eight metabotropic glutamate
receptors (mGluR1–mGluR8), and six orphan recep-
tors, including the recently characterized GPRC6A (7,
68). These receptors appear to share an evolutionary
thread with CaR based on their common functional
origins as nutrient/salinity sensors (15, 30). The CaR is
allosterically modulated by extracellular amino acids
(15). Conversely, other members of this family that are
regarded as amino acid sensors, such as certain
mGluRs, GABA
receptors, and GPRC6A, are modulat-
ed by extracellular Ca
(44). GPRC6A has 34% amino
acid sequence identity with CaR (68) and is activated
by relatively high extracellular [Ca
] (5–10 mM) (44).
This receptor has been suggested to serve as a sensor
for Ca
in bone (44), a tissue where local extremes in
external [Ca
] are believed to occur during the bone
remodeling process.
Many other cell surface proteins are susceptible to
physiological fluctuations in external [Ca
] (recently
reviewed in Ref. 32). These include gap-junction
hemichannels (64), which can open in response to a
modest (~200 M) decrease in external [Ca
], and the
receptor Notch, which may sense external [Ca
] to
drive the establishment of right-left symmetry during
embryogenesis (50, 51). A distinct Ca
-sensing recep-
tor known as CAS has been recently described in
plants (59). In addition, a number of ion channels alter
their open probability depending on the local extracel-
lular Ca
, including the proton-gated cation channels
channels, and other nonse-
lective channels found in neuronal tissue (32).
PHYSIOLOGY • Volume 22 • October 2007 •
transport of Ca
buffers (e.g., HCO
, PO
) would also
be expected to influence the free [Ca
] in the intersti-
tium. Ca
taken up into endocytic vesicles could con-
ceivably impact the local extracellular [Ca
] (23).
Finally, secretory vesicles are known to contain high
concentrations of Ca
and other divalent cations (Zn
), and synchronous secretory activity could in
principle lead to rapid increases in extracellular diva-
lents (29, 48). Gray et al. recently proposed that libera-
tion of these metals from vesicles of insulin-secreting
cells may constitute a means of communication
between cells (29) via sensors for extracellular Ca
, as
described in the following section.
FIGURE 4. Extracellular [Ca
] becomes elevated adjacent to
stimulated cells due to Ca
Direct measurements of Ca
extrusion from pancreatic acinar cells per-
formed by Tepikin and colleagues (62) using the “droplet technique”
demonstrates that large quantities of Ca
are exported from stimulated
cells. The total drop in cellular Ca
following acetylcholine (ACh) treat-
ment was estimated to be about 0.7 mM over 2–5 min (modified with
permission from Ref. 62). A: photomicrograph of fluid micro-droplet con-
taining a single mouse pancreatic acinar cell. The cell was loaded with the
indicator fura-2, whereas the droplet contained a second Ca
fluo-3. At right is seen the pipette tip, used for iontophoretic delivery of
agonists. B: simultaneous measurement of free intracellular Ca
tration ([Ca
) in a single acinar cell and extracellular Ca
) in the
droplet following challenge with 20 nM ACh.
Intercellular Communication
Via Ca
Our laboratory demonstrated some years ago in a
proof-of-concept study using a co-culture model sys-
tem that it is possible for CaR to detect extracellular
fluctuations in [Ca
] that occur secondary to intracel-
lular Ca
signaling events (34, 65). This opened up the
prospect that Ca
might function as a paracrine
messenger, used, for example, to communicate infor-
mation about the signaling status of a neighboring cell
or to integrate or reinforce signals in multicellular
ensembles. We further provided evidence for a varia-
tion on this theme, whereby exported Ca
can activate
CaR expressed on the same cell in an autocrine fash-
ion (19). Caroppo et al. (13) later showed a physiolog-
ical role of this third messenger signaling system in the
intact gastric mucosa. These investigators took advan-
tage of information gained from their previous extra-
cellular microelectrode studies aimed at measuring
the profile of the extracellular Ca
signal” in the
apical and basolateral microdomains following
cholinergic stimulation (see above) (13). Remarkably,
reproducing this physiological pattern of extracellular
] variation was able to elicit changes in pepsino-
gen and alkaline secretion from the tissue (14), and
more recently this third messenger activity has been
linked to changes in water transport (24) in the same
model system. The CaR, which is expressed apically in
the amphibian oxyntic cell, is involved in detecting the
extracellular [Ca
] elevation that occurs in the lumi-
nal compartment of the gastric gland, although it
appears that another entity may be responsible for
sensing the basolateral decrease in [Ca
] (14). These
intriguing data are suggestive of a novel mode of Ca
signaling that takes advantage of extracellular, rather
than intracellular, changes in [Ca
], but it remains to
be seen whether this process occurs in other tissues.
Other Second Messengers as “Third
Are there other hydrophilic signaling molecules that
are exported by cells to inform neighboring cells of
their signaling or metabolic status? Cyclic GMP, the
second messenger generated by either atrial natri-
uretic peptide or nitric oxide gas via guanylate
cyclases, is vigorously exported from many cell types
in quantities that surpass that of cAMP (4, 54). This
widespread phenomenon is mediated by many of the
same MRP family members (e.g., MRP4, MRP5,
MRP8) known to transport cAMP, as well as the
organic anion transporter OAT1 (69). Diverse biologi-
cal actions of extracellular cGMP have been described
in brain and kidney [recently reviewed by Sager (54)],
but as is the case for extracellular cAMP, specific
molecular receptors for cGMP in mammalian cells
have yet to be identified.
Isolated reports of extracellular accumulation of
inositol 1,4,5 trisphosphate (InsP
) following choliner-
gic stimulation as measured using microdialysis tech-
niques in brain have also appeared (40, 52). Roberts et
al. found that several inositol phosphate metabolites
of InsP
appeared (in addition to InsP
) in the intersti-
tial space under these conditions, although it is uncer-
tain whether the appearance of the additional inositol
derivatives reflects metabolism of extracellular InsP
or a separate transport process (52). It is not known
whether InsP
egress is a widespread phenomenon or
whether it has any functional significance.
Second messengers are the cellular currency of infor-
mation transfer. However, the generation of cAMP from
ATP and the energy required to maintain the gradients
that permit Ca
signaling to take place come at a cer-
tain energetic cost. Thus it is attractive to imagine that
multicellular organisms might capitalize on fluctua-
tions in extracellular second messengers to expand the
informational content of the intracellular signal trans-
duction process. The concept of the interstitial
microdomain as a specialized signaling compartment
is in its infancy, however. There is still much to learn
about how and when the local concentrations of cAMP
and Ca
change in this space and what the physiologi-
cal consequences of these fluctuations are. The devel-
opment of practical methods for probing the profile of
such extracellular “signals” will be an important first
step to understanding whether this constitutes a gener-
alized device to extend the scope and range of second
messenger molecules to a domain outside the cell.
We are grateful to Dr. Kees Jalink of the Netherlands
Cancer Institute for graciously providing us with the Epac-
based FRET sensor used for imaging intracellular cAMP
and to our colleague Prof. Silvana Curci for reading the
Support for the work conducted in the Hofer laboratory
was provided by the Medical Research Service of the
Department of Veteran’s Affairs and the Brigham Surgical
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PHYSIOLOGY • Volume 22 • October 2007 •
PHYSIOLOGY • Volume 22 • October 2007 •
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PHYSIOLOGY • Volume 22 • October 2007 •
... Specifically, cAMP inhibits cell growth at high concentrations [8]. cAMP also relaxes smooth muscle cells [9]. ...
... In addition to hydrolysis by PDEs, intracellular cAMP is removed from cells by efficient export to the extracellular space [12][13][14]. Because extracellular cAMP has direct and indirect autocrine/paracrine effects [9], egress of cAMP serves to broaden the signaling roles of cAMP. ...
Full-text available
Autocrine/paracrine factors generated in response to 17β-estradiol (E2) within the fallopian tube (FT) facilitate fertilization and early embryo development for implantation. Since cyclic AMP (cAMP) plays a key role in reproduction, regulation of its synthesis by E2 may be of biological/pathophysiological relevance. Herein, we investigated whether cAMP production in FT cells (FTCs) is regulated by E2 and environmental estrogens (EE’s; xenoestrogens and phytoestrogens). Under basal conditions, low levels of extracellular cAMP were detectable in bovine FTCs (epithelial cells and fibroblasts; 1:1 ratio). Treatment of FTCs with forskolin (AC; adenylyl cyclase activator), isoproterenol (β-adrenoceptor agonist) and IBMX (phosphodiesterase (PDE) inhibitor) dramatically (>10 fold) increased cAMP; whereas LRE1 (sAC; soluble AC inhibitor) and 2’,5’-dideoxyadenosine (DDA; transmembrane AC (tmAC)) inhibitor decreased cAMP. Comparable changes in basal and stimulated intracellular cAMP were also observed. Ro-20-1724 (PDE-IV inhibitor), but not milrinone (PDE-III inhibitor) nor mmIBMX (PDE-I inhibitor), augmented forskolin-stimulated cAMP levels, suggesting that PDE-IV dominates in FTCs. E2 increased cAMP levels and CREB phosphorylation in FTCs, and these effects were mimicked by EE’s (genistein, 4-hydroxy-2’,4’,6’-trichlorobiphenyl, 4-hydroxy-2’,4’,6’-dichlorobiphenyl). Moreover, the effects of E2 and EE were blocked by the tmAC inhibitor DDA, but not by the ERα/β antagonist ICI182780. Moreover, BAPTA-AM (intracellular-Ca2+ chelator) abrogated the effects of E2, but not genistein, on cAMP suggesting differential involvement of Ca2+. Treatment with non-permeable E2-BSA induced cAMP levels and CREB-phosphorylation; moreover, the stimulatory effects of E2 and EEs on cAMP were blocked by G15, a G protein-coupled estrogen receptor (GPER) antagonist. E2 and IBMX induced cAMP formation was inhibited by LRE1 and DDA suggesting involvement of both tmAC and sAC. Our results provide the first evidence that in FTCs, E2 and EE’s stimulate cAMP synthesis via GPER. Exposure of the FT to EE’s and PDE inhibitors may result in abnormal non-cyclic induction of cAMP levels which may induce deleterious effects on reproduction.
... It has been reported that the enhanced osteogenic ability of CO 3 Ap is caused by the release of calcium ions, which can bind to the cell membrane via G protein-coupled receptors to activate the extracellular signal-related kinase pathway [22,23]. Furthermore, a much higher osteoconductivity of CO 3 Ap was reported compared to that of b-TCP [10]. ...
Full-text available
Objective: This study aimed to histologically compare periodontal regeneration of one-wall intrabony defects treated with open flap debridement, β-tricalcium phosphate (β-TCP), and carbonate apatite (CO3Ap) in dogs. Methods: The mandibular third premolars of four beagle dogs were extracted. Twelve weeks after the extraction, a one-wall bone defect of 4 mm × 5 mm (mesio-distal width × depth) was created on the distal side of the mandibular second premolar and mesial side of the fourth premolar. Each defect was randomly allocated to open flap debridement (control group), periodontal regeneration utilizing β-TCP, or CO3Ap. Eight weeks after the surgery, histologic and histometric analyses were performed. Results: No ankylosis, infection, or acute inflammation was observed at any of the experimental sites. Newly formed bone and cementum were observed in all experimental groups. The mineral apposition rate of the alveolar bone crest was higher in the CO3Ap group than in the control and β-TCP groups. The ratio of the new bone area was significantly higher in the CO3Ap group than in the control group (P < 0.05). The bone contact percentage of the residual granules was significantly higher in the CO3Ap group than in the β-TCP group (P < 0.05). Conclusion: Although this study has limitations, the findings revealed the safety and efficacy of CO3Ap for periodontal regeneration in one-wall intrabony defects in dogs, and CO3Ap has a better ability to integrate with bone than β-TCP.
... Unlike other externally-imposed challenges, the subsequent and spontaneous relaxation of the ECs from the SRBW mechanostimulation to their ground state following the sonochallenge phase is accompanied not just by a restoration of the endothelial barrier but also an enhancement in its capacity-without requiring additional stimuli. Again, we observe Ca 2+ signalling to be central in this subsequent sonotransformation phase, albeit through the release of another second messenger, cAMP [93][94][95]. That the SRBW mechanostimulation uniquely progresses spontaneously from the initial insult (sonochallenge) phase to initiate cAMP-mediated barrier recovery in the sonotransformation phase is likely due to its ability to increase intracellular Ca 2+ levels beyond a particular threshold to trigger its storage in the ER for subsequent release back into the extracellular millieu, which relies on ATP conversion into cAMP to fuel the process. ...
The endothelial junction plays a central role in regulating intravascular and interstitial tissue permeability. The ability to manipulate its integrity therefore not only facilitates an improved understanding of its underlying molecular mechanisms but also provides insight into potential therapeutic solutions. Herein, we explore the effects of short-duration nanometer-amplitude MHz-order mechanostimulation on interendothelial junction stability and hence the barrier capacity of endothelial monolayers. Following an initial transient in which the endothelial barrier is permeabilised due to Rho-ROCK-activated actin stress fibre formation and junction disruption typical of a cell's response to insults, we observe, quite uniquely, the integrity of the endothelial barrier to not only spontaneously recover but also to be enhanced considerably-without the need for additional stimuli or intervention. Central to this peculiar biphasic response, which has not been observed with other stimuli to date, is the role of second messenger calcium and cyclic adenosine monophosphate (cAMP) signalling. We show that intracellular Ca2+, modulated by the high frequency excitation, is responsible for activating reorganisation of the actin cytoskeleton in the barrier recovery phase, in which circumferential actin bundles are formed to stabilise the adherens junctions via a cAMP-mediated Epac1-Rap1 pathway. Despite the short-duration stimulation (8 min), the approximate 4-fold enhancement in the transendothelial electrical resistance (TEER) of endothelial cells from different tissue sources, and the corresponding reduction in paracellular permeability, was found to persist over hours. The effect can further be extended through multiple treatments without resulting in hyperpermeabilisation of the barrier, as found with prolonged use of chemical stimuli, through which only 1.1- to 1.2-fold improvement in TEER has been reported. Such an ability to regulate and enhance endothelial barrier capacity is particularly useful in the development of in vitro barrier models that more closely resemble their in vivo counterparts.
... Genetic variation in both genes, FOXP2 and PDE4D, was previously associated with schizophrenia [82][83][84][85]. PDE4D is involved in cAMP degradation [56], a signal transduction molecule influencing a broad range of cellular functions [86,87], whereas FOXP2 encodes for the transcription factor forkhead box P2, which has been widely linked to speech and language development [57][58][59]. Verbal fluency deficits and disorganized speech are often observed in schizophrenia patients [88,89], and may be related to altered FOXP2 expression and/or functioning [90]. ...
Full-text available
The midbrain is an extensively studied brain region in schizophrenia, in view of its reported dopamine pathophysiology and neuroimmune changes associated with this disease. Besides the dopaminergic system, the midbrain contains other cell types that may be involved in schizophrenia pathophysiology. The neurovascular hypothesis of schizophrenia postulates that both the neurovasculature structure and the functioning of the blood-brain barrier (BBB) are compromised in schizophrenia. In the present study, potential alteration in the BBB of patients with schizophrenia was investigated by single-nucleus RNA sequencing of post-mortem midbrain tissue (15 schizophrenia cases and 14 matched controls). We did not identify changes in the relative abundance of the major BBB cell types, nor in the sub-populations, associated with schizophrenia. However, we identified 14 differentially expressed genes in the cells of the BBB in schizophrenia as compared to controls, including genes that have previously been related to schizophrenia, such as FOXP2 and PDE4D. These transcriptional changes were limited to the ependymal cells and pericytes, suggesting that the cells of the BBB are not broadly affected in schizophrenia.
... It has been reported that the enhanced osteogenic ability of CO 3 Ap is caused by the release of calcium ions, which can bind to the cell membrane via G protein-coupled receptors to activate the extracellular signal-related kinase pathway [22,23]. Furthermore, a much higher osteoconductivity of CO 3 Ap was reported compared to that of b-TCP [10]. ...
Full-text available
Objective : This study aimed to histologically compare periodontal regeneration of one-wall intrabony defects treated with open flap debridement, β-tricalcium phosphate (β-TCP), and CO3Ap in dogs. Materials and Methods : The mandibular third premolars of four beagle dogs were extracted. Twelve weeks after the extraction, a one-wall bone defect of 4 mm × 5 mm (mesio-distal width × depth) was created on the distal side of the mandibular second premolar and mesial side of fourth premolar. Each defect was randomly subjected to open flap debridement only (control group), β-TCP, or CO3Ap treatment. Eight weeks after the surgery, histologic and histometric analyses were performed. Results : No ankylosis, infection, or acute inflammation was observed at any of the experimental sites. Newly formed bone and cementum were observed in all experimental groups. The ratio of the new bone area was significantly higher in the CO3Ap group than in the control group ( P < 0.05). The mineral apposition rate of the alveolar bone crest was higher in the CO3Ap group than in the control and β-TCP groups. The bone contact percentage of the residual granules was significantly higher in the CO3Ap group than in the β-TCP group ( P < 0.05). Conclusion : These findings indicate the safety and efficacy of CO3Ap for periodontal regeneration in one-wall intrabony defects in dogs, and CO3Ap is more integrated with bone than β-TCP. Clinical relevance : CO3Ap is compatible with the surrounding bone and provides favorable results for periodontal regeneration in intrabony defects.
... Genetic variation in both genes, FOXP2 and PDE4D, was previously associated with schizophrenia [82][83][84][85]. PDE4D is involved in cAMP degradation [56], a signal transduction molecule influencing a broad range of cellular functions [86,87], whereas FOXP2 encodes for the transcription factor forkhead box P2, which has been widely linked to speech and language development [57][58][59]. Verbal fluency deficits and disorganized speech are often observed in schizophrenia patients [88,89], and may be related to altered FOXP2 expression and/or functioning [90]. ...
Full-text available
The midbrain is an important brain region for the study of schizophrenia in view of its reported dopamine pathophysiology and observed neuroimmune changes associated with schizophrenia. Besides the dopaminergic system, the midbrain contains other cell types that may be involved in schizophrenia pathophysiology. The neurovascular hypothesis of schizophrenia postulates that both the neurovasculature structure and the functioning of the blood-brain barrier (BBB) are compromised in schizophrenia. In the present study, potential alteration in the BBB of patients with schizophrenia was investigated by single-nucleus RNA sequencing of post-mortem midbrain tissue (14 schizophrenia cases and 15 matched controls). We did not identify changes in the relative abundance of the major BBB cell types, nor in the sub-populations, associated with schizophrenia. However, we identified 14 differentially expressed genes in the cells of the BBB in schizophrenia as compared to controls, including genes that have previously been related to schizophrenia, such as FOXP2 and PDE4D . These transcriptional changes associated with schizophrenia were limited to the ependymal cells and pericytes. This schizophrenia cohort was previously stratified into “high inflammation” and “low inflammation” cases, based on cortical inflammation-related transcripts. We detected a sub-population of protoplasmic astrocytes enriched in the high inflammation schizophrenia subgroup. Genes more abundantly expressed in these schizophrenia-related protoplasmic astrocytes were associated with glutamatergic synaptic function rather than with inflammation. In summary, transcriptional changes in the cells of the BBB in schizophrenia are limited and specific. In addition, inflammation may be affecting the function of astrocytes in a subgroup of schizophrenia patients, and thereby contribute to schizophrenia pathophysiology.
... Unlike metabolic pathways that consists of enzymatic reactions where substrates are converted to products, signaling pathways consists of signal transduction cascades controlled by sensors, transducers and actuators [56]. Signal cascades ( Figure 2) can be divided in three parts: (i) signaling molecules binding to receptors (in many cases transmembrane proteins that sense extracellular molecules) and initiation of the cascade; (ii) signal transduction driven by interactions between the proteins within the pathway (using mechanisms such as phosphorylation, ubiquitination, cellular translocation [57][58][59][60]), and small signal carrying molecules such as cyclic AMP (cAMP) and ions such as sodium, magnesium and calcium [61,62]; and (iii) induction and repression of genes, or activation and inactivation of proteins and enzymes (the end-outcome of the signal cascade). In addition to their specific functions, many signaling networks interact in a phenomenon known as cross-talk (signal transduction between signaling pathways [56]). ...
Full-text available
Extension of the substrate range is among one of the metabolic engineering goals for microorganisms used in biotechnological processes because it enables the use of a wide range of raw materials as substrates. One of the most prominent examples is the engineering of baker’s yeast Saccharomyces cerevisiae for the utilization of d-xylose, a five-carbon sugar found in high abundance in lignocellulosic biomass and a key substrate to achieve good process economy in chemical production from renewable and non-edible plant feedstocks. Despite many excellent engineering strategies that have allowed recombinant S. cerevisiae to ferment d-xylose to ethanol at high yields, the consumption rate of d-xylose is still significantly lower than that of its preferred sugar d-glucose. In mixed d-glucose/d-xylose cultivations, d-xylose is only utilized after d-glucose depletion, which leads to prolonged process times and added costs. Due to this limitation, the response on d-xylose in the native sugar signaling pathways has emerged as a promising next-level engineering target. Here we review the current status of the knowledge of the response of S. cerevisiae signaling pathways to d-xylose. To do this, we first summarize the response of the native sensing and signaling pathways in S. cerevisiae to d-glucose (the preferred sugar of the yeast). Using the d-glucose case as a point of reference, we then proceed to discuss the known signaling response to d-xylose in S. cerevisiae and current attempts of improving the response by signaling engineering using native targets and synthetic (non-native) regulatory circuits.
... Most metals are distributed in blood cells but a few acts as messengers outside the blood cells. For example, Ca is the second messenger outside the blood cells and can bind to troponin (Hofer and Lefkimmiatis 2007). ...
Full-text available
It is important but remains unclear whether ethylenediaminetetraacetic acid (EDTA) and sodium heparin anticoagulants have different impacts on the levels of various metals in peripheral blood after long-term frozen storage. The concentrations of 22 metals (Al, As, Ba, Ca, Cd, Cr, Co, Cu, Mn, Mg, Mo, Ni, Fe, Pb, Rb, Se, Sn, Sb, Sr, Ti, V, Zn) in whole blood, blood cells and plasma from 22 healthy participants were determined twice, 18 months apart, using inductively coupled plasma mass spectrometry (ICP-MS). The mean percentage error (MPE) and intraclass correlation coefficient (ICC) were calculated to evaluate the impact of the anticoagulants and long-term frozen storage on metal concentrations, respectively. The concentrations of Sb and Ba in whole blood, blood cells and plasma were significantly altered by EDTA and sodium heparin at two measurement timepoints (P < 0.05 and MPE > 80%). In EDTA tubes, the Ti and Ni concentrations in blood cells were changed significantly; and in heparin tubes, the concentrations of Ni and Mo in blood cells and Sb in plasma were also altered (P < 0.05 and MPE > 80%). The ICCs of 11 metals in whole blood, 15 metals in blood cells and 16 metals in plasma remained unchanged in EDTA tubes, and 16 metals in whole blood, 15 metals in blood cells and 17 metals in plasma remained unchanged in heparin tubes (ICC > 0.40). Our study suggested the use of EDTA tubes to determine Sb concentrations in peripheral blood and heparin tubes to determine Ba concentrations. Additionally, heparin tubes may be more suited for determining multiple metal concentrations in whole blood, whereas for blood cells and plasma either EDTA or heparin tubes could be used.
... Perhaps the most interesting of the 69 EOF-exclusive metabolites identified (Fig. 3A) was adenosine 3′-5′-cyclic monophosphate (cAMP)-whose supplementation to culture media is required to induce stromal cell decidualization in vitro (74). While the mechanisms of extracellular cAMP secretion, via ATP-binding cassette C (ABCC) transporters, are well characterized in other systems (75,76), the potential paracrine function of cAMP in endometrial epithelial-stromal cross-talk has yet to be established. Intriguingly, however, EEOs from all three donors expressed eight ABCCs, including ABCC4, whose dysregulation in vivo is associated with endometriosis (77). ...
Significance The ability to robustly isolate intraorganoid fluid in a high-throughput capacity, using human endometrial epithelia as a model, circumvents several constraints typically associated with obtaining biological fluids, both in vivo and in vitro. This approach also allows for direct tandem genomics, transcriptomics, proteomics, and functional metabolomics. In addition to enhancing our understanding of uterine fluid composition regulation, this innovation is directly and readily transferrable to wider fields of cell and organoid biology, with implications for personalized, or precision, medicine approach development.
‘Biostimulants’ appeared as the boon to the sustainable agriculture system which are actually versatile products (natural and chemically synthesized) and/or organisms (beneficial microbes, free-living or rhizospheric or endosymbiotic) which significantly potentiates the process of agronomic trait improvement in plants without compromising the final yield, quality and ecological balance. In this emerging field of agriculture, the fungal endophytes have been considered as potential “biostimulants” that could facilitate commercial bio-prospecting of high value secondary metabolites from higher and lower groups of medicinal plants in the most sustainable, mutualistic. Recently, cultivation of medicinal and aromatic crops has secured special status in agriculture and conservation system. Globally, the accelerated demand for complementary and alternative medicine has widened economic prospects of farmers and huge profitability could be predicted for elite chemotypes (high metabolite yielding varieties suitable for industrial harvest). The natural co-existence of endophytes, and their cross talk with host plant regulating secondary metabolism, stress reversal and defense has been investigated extensively both in wild and in-vitro condition. In this chapter we have highlighted fungal endophytes and their role as ‘microbial consortia’ in mutual symbiosis with medicinal crops (host), which constitutively triggered higher accumulation of expensive biopharmaceuticals, growth promotion, biotic and abiotic stress tolerance. Moreover, streamlined discussion has been provided for mechanism of action, modulation and signal transduction, biotechnological advancement and futuristic manipulative strategies to aid sustainable agriculture system.
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The intracellular free Ca2+ concentration ([free Ca2+]i) was measured simultaneously with the Ca2+ extrusion from single isolated mouse pancreatic acinar cells placed in a microdroplet of extracellular solution using the fluorescent probes fura-2 and fluo-3. The extracellular solution had a low total calcium concentration (15-35 microM), and acetylcholine (ACh), applied by microionophoresis, therefore only evoked a transient elevation of [free Ca2+]i lasting about 2-5 min. The initial sharp rise in [free Ca2+]i from about 100 nM toward 0.5-1 microM was followed within seconds by an increase in the total calcium concentration in the microdroplet solution ([Ca]o). The rate of this rise of [Ca]o was dependent on the [free Ca2+]i elevation, and as [free Ca2+]i gradually decreased Ca2+ extrusion declined with the same time course. Ca2+ extrusion following ACh stimulation was not influenced by removal of all Na+ in the microdroplet solution indicating that the Ca2+ extrusion is not mediated by Na(+)-Ca2+ exchange but by the Ca2+ pump. The amount of Ca2+ extruded during the ACh-evoked transient rise in [free Ca2+]i corresponded to a decrease in the total intracellular Ca concentration of about 0.7 mM which is close to previously reported values (0.5-1 mM) for the total concentration of mobilizable calcium in these cells. Our results therefore demonstrate directly the ability of the Ca2+ pump to rapidly remove the large amount of Ca2+ released from the intracellular pools during receptor activation.
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Both 3':5' cyclic adenosine monophosphate (cAMP) and 3':5' cyclic guanosine monophosphate (cGMP) stimulated colony-stimulating factor 1 (CSF-1)-dependent colony formation by murine two-signal-dependent progenitors without influencing colony formation by committed CSF-1-responsive progenitors. The stimulatory effect was optimal at 10(-9) M and did not diminish with increasing concentrations of the cyclic nucleotides. The membrane-permeating analogs dibutyryl cAMP and 8-Br-cGMP similarly augmented colony formation by the transitional progenitors at 10(-9) M; however, with increasing concentration, enhancement diminished with eventual inhibition of total colony formation at micromolar concentrations. Stimulation by the two cyclic nucleotides was mutually incompatible. The results indicate that physiological levels of extracellular cyclic nucleotides may significantly influence myelopoiesis. Furthermore, the results introduce the interesting possibility that stimulation, unlike inhibition, may be initiated through an extracytoplasmic mechanism that does not require direct activation of cytoplasmic cyclic nucleotide-dependent protein kinases.
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Ca2+ extrusion was measured simultaneously with the free intracellular Ca2+ concentration ([Ca2+]i) from single pancreatic acinar cells placed in microdroplets of extracellular solution (Tepikin, A. V., Voronina, S. G., Gallacher, D. V., and Petersen, O. H. (1992) J. Biol. Chem. 267, 3569-3572). Submaximal stimulation with cholecystokinin usually evoked discrete cytosolic Ca2+ spikes and each of these spikes was associated with a discrete and virtually synchronous pulse of Ca2+ extrusion into the extracellular microdroplet solution. When ACh evoked repetitive discrete [Ca2+]i spikes, each spike was also accompanied by a discrete pulse of Ca2+ extrusion. The velocity of Ca2+ extrusion oscillated with a time course similar to that of [Ca2+]i. The extracellular solution in our experiments had a low total calcium concentration (15-35 microM) and only a limited number of [Ca2+]i spikes (2-8) could be evoked. The magnitudes of the [Ca2+]i spikes and the amounts of Ca2+ extruded during each spike gradually decreased in each experiment. During the first cholecystokinin-evoked cytosolic Ca2+ spike the Ca2+ extrusion corresponded to a loss of 15-70% (mean value 39% +/- 12) of the mobilizable cellular calcium pool. The substantial pulsatile Ca2+ extrusion occurring synchronously with the receptor-activated cytosolic Ca2+ spikes is therefore an important element in repetitively bringing back [Ca2+]i to the resting level.
We isolated osteoblastic cell lines from wild-type (CasR +/+) and receptor null (CasR −/−) mice to investigate whetherCasR is present in osteoblasts and accounts for their responses to extracellular cations. Osteoblasts from bothCasR +/+ and CasR −/−mice displayed an initial period of cell replication followed by a culture duration-dependent increase in alkaline phosphatase activity, expression of osteocalcin, and mineralization of extracellular matrix. In addition, a panel of extracellular cations, including aluminum and the CasR agonists gadolinium and calcium, stimulated DNA synthesis, activated a transfected serum response element-luciferase reporter construct, and inhibited agonist-induced cAMP in CasR −/− osteoblasts. The functional responses to these cations were identical inCasR +/+ and CasR −/−osteoblasts. Thus, the absence of CasR alters neither the maturational profile of isolated osteoblast cultures nor their in vitro responses to extracellular cations. In addition,CasR transcripts could not be detected by reverse transcription-polymerase chain reaction with mouse specific primers in either CasR +/+ orCasR −/− osteoblasts, and immunoblot analysis with a CasR-specific antibody was negative forCasR protein expression in osteoblasts. The presence of a cation-sensing response in osteoblasts fromCasR −/− mice indicates the existence of a novel osteoblastic extracellular cation-sensing mechanism.
The MRP family is comprised of nine related ABC transporters that are able to transport structurally diverse lipophilic anions and function as drug efflux pumps. Investigations of this family have provided insights not only into cellular resistance mechanisms associated with natural product chemotherapeutic agents, antifolates and nucleotide analogs, but also into factors that influence drug distribution in the body, membrane systems that are involved in the extrusion of reduced folates, cysteinyl leukotrienes and bile acids, and the molecular basis of two hereditary conditions in humans. The review will describe the biochemical properties, drug resistance activities and potential in vivo functions of these unusual pumps.
The effects of dopaminergic agonists and antagonists have been studied in dispersed bovine parathyroid cells. Dopaminergic agonists caused a transient 20- to 40-fold increase in cellular cyclic AMP and a 2- to 3-fold increase in parathyroid hormone release. Dose-response relationships were similar for cyclic AMP accumulation and hormone release, whether studied by increasing agonist concentration or by increasing concentration of antagonist with constant agonist. The effects on the dopamine receptor could be differentiated from those of the previously characterized beta-adrenergic receptor by specific inhibitors. These results appear to represent proof with a homogeneous cell population that dopaminergic receptors linked to adenylate cyclase can regulate a secretory process mediated by cyclic AMP. This system should be useful in further studies on dopamine receptors and should provide a valid tool for determining interactions of radiolabeled ligands with such receptors.
Small doses (10-150 microgram; 3-45 nmol) of glucagon caused a dose-dependent increase in plasma adenosine 3':5'-cyclic monophosphate (cyclic AMP) concentration when injected into man. Infusion of glucagon (75 ng min-1 kg-1) for 2 h into normal subjects resulted in an initial increase in plasma cyclic AMP concentration, then a decline despite continuation of the hormone infusion and maintenance of high concentrations of circulating immunoreactive glucagon. When an injection of glucagon was given at the termination of such an infusion, the subsequent increase in plasma cyclic AMP concentration was markedly reduced when compared to that observed after a control injection which had not been preceded by a glucagon infusion. When the glucagon was injected at the end of an infusion of 1000 MRC units of bovine parathyroid hormone (BPTH) over 2 h, the plasma cyclic AMP response was normal. Conversely, after infusion of glucagon the response to injected BPTH was normal. This impairment of response was therefore specific to the hormone that had been administered and was not due to altered metabolism of circulating cyclic AMP. This phenomenon may be important in the regulation of the hormonal response by the target tissue.
The phosphorylation of the cardiac sodium channel by adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase A leads to its inactivation. It was shown that extracellular cAMP can also modulate the sodium channel of rat, guinea pig, and frog ventricular myocytes in a rapid (less than 50 milliseconds), reversible, and dose-dependent manner. The decrease in the sodium current was accompanied by a 10- to 15-millivolt shift in the steady-state availability of the sodium channel toward more negative potentials and was inhibited by guanosine-5'-O-(2-thiodiphosphate) or pertussis toxin, suggesting that the extracellular modulation of the sodium channel by cAMP is mediated by a membrane-delimited mechanism that includes a pertussis toxin-sensitive G protein.
Simultaneous optical measurements of extra- and intracellular Ca2+ concentrations were carried out on isolated snail neurons injected iontophoretically with Ca2+. The fluorescent indicator Fura-2 was used to measure intracellular concentration of free Ca, and the absorbant indicator Antipyrylazo III to measure changes in extracellular calcium concentration in the micro-chamber containing the cell. The velocity of Ca2+ extrusion from a single cell has been shown to be in accordance with the level of free Ca in the neuronal cytoplasm. After an increase in intracellular free Ca by iontophoretic injection from a microeletrode to 0.2-0.5 microM, the velocity of Ca2+ extrusion from the neuron was approximately 0.3-4.6 microM/sec per cell volume. During caffeine-induced calcium-dependent calcium release of Ca2+ from intracellular stores a stimulation of calcium extrusion took place, reaching the velocity of 5.0 microM/sec per cell volume.